The present invention relates generally to the stem cell field and, more particularly, to employing stem cells in the development of rat cell lines and animals.
Propagation of Spermatogonial Stem Cell Lines
The ability to conditionally induce the development of stem cell lines through the process of spermatogenesis in vitro for the production of gametes would provide a long-sought-after technology for biomedical research, particularly if such protocols could be established for a variety of species. The discovery that stem cells residing within fractions of dissociated mouse and rat testis cells maintain their ability to regenerate spermatogenesis in testes of recipient mice was essential to establishing such culture systems. See Brinster et al., Proc Natl Acad Sci USA 1994; 91:11303-11307; Brinster et al., Proc Natl Acad Sci USA 1994; 91:11298-11302; Clouthier et al., Nature 1996; 381:418-421; Kanatsu-Shinohara et al., Biol Reprod 2003; 69:612-616; and Nagano et al., Tissue Cell 1998; 30:389-397. The ability to isolate and experimentally manipulate these stem cells has opened new doors for research on spermatozoan development, assisted reproduction, cellular therapy and genetics. See Nagano et al., Biol Reprod 1999; 60:1429-1436; Mahato et al., Endocrinology 2000; 141:1273-1276; Mahato et al., Mol Cell Endocrinol 2001; 178:57-63; Ogawa et al., Nat Med 2000; 6:29-34; Shinohara et al., Proc Natl Acad Sci USA 2006; 103:13624-13628; Zhang et al., J Cell Physiol 2007; 211:149-158; Kazuki et al., Gene Ther 2008; 15:617-624; Kanatsu-Shinohara et al., Cell 2004; 119:1001-1012; Kanatsu-Shinohara et al., Proc Natl Acad Sci USA 2006; 103:8018-8023; and Nagano et al., Proc Natl Acad Sci USA 2001; 98:13090-13095. In view of this potential, protocols for isolating, propagating and genetically modifying fully functional rat spermatogonial stem cells in culture have been established. See Ryu et al., Dev Biol 2004; 274:158-170; Hamra et al., Dev Biol 2004; 269:393-410; Hamra et al., Proc Natl Acad Sci USA 2002; 99:14931-14936; Hamra et al., Methods Mol Biol 2008; 450:163-179; Hamra et al., Proc Natl Acad Sci USA 2005; 102:17430-17435; Ryu et al., Proc Natl Acad Sci USA 2005; 102:14302-14307; Orwig et al., Biol Reprod 2002; 67:874-879; and Kanatsu-Shinohara et al., Biol Reprod 2008. The rat was chosen as a species for these studies due to its popularity as a laboratory animal model for the study of human health and disease, and due to the lack of protocols for genetically modifying the rat germline using clonally expanded stem cells from culture. See Hamra et al., Proc Natl Acad Sci USA 2002; 99:14931-14936 Considering the many potential applications of the laboratory rat as a research model, a cost-effective and easy-to-prepare culture medium was sought in this study for the derivation and continuous proliferation of primary rat spermatogonial stem cell lines in vitro.
In respect of this goal, media previously reported for long-term proliferation of rodent spermatogonial stem cells in vitro represent clear methodological advances for studies on the biology and applications of spermatogonia. See Kanatsu-Shinohara et al., Biol Reprod 2003; 69:612-616; Hamra et al., Proc Natl Acad Sci USA 2005; Ryu et al., Proc Natl Acad Sci USA 2005; 102:14302-14307; Kubota et al., Proc Natl Acad Sci USA 2004; 101:16489-16494; and Kanatsu-Shinohara et al., Biol Reprod 2005; 72:985-991. However, such media are relatively complex, expensive, time-consuming to prepare, plus are most effective when applied in combination with feeder layers of fibroblasts. See id. For example, the medium originally reported by Shinohara and colleagues for the successful derivation and long-term cultivation of germline stem cells from postnatal mouse testes was a pivotal breakthrough in spermatogonial research. See Kanatsu-Shinohara et al., Biol Reprod 2003; 69:612-616. However, Shinohara's medium is based on the proprietary, StemPro-34 medium, plus 24 individually added components, including small molecules, fetal bovine serum and a mixture of polypeptide growth factors. Serum-free derivatives of Shinohara's medium have since been formulated for spermatogonial culture, in which the serum has been replaced by the supplement, B-27. See Hamra et al., Proc Natl Acad Sci USA 2005; 102:17430-17435 and Kanatsu-Shinohara et al., Biol Reprod 2005; 72:985-991. Upon inspection of components within B-27 supplement we postulated that it could be used together with key growth factors in a commonly applied nutrient mixture to formulate a more efficient spermatogonial culture medium.
Sterile Testes Complementation
Currently, specific causes of infertility in men remain a mystery in 40-60% of cases. See Bhasin et al., J Clin Endocrinol Metab 79, 1525-9 (1994); Sadeghi-Nejad et al., Urol J 4, 192-206 (2007); and Matzuk et al., Nat Med 14, 1197-213 (2008). In total, >5% of the male population is infertile, and >1% of all males are inflicted with a severe defect in sperm production termed azoospermia. See Bhasin et al., J Clin Endocrinol Metab 79, 1525-9 (1994); Sadeghi-Nejad et al., Urol J 4, 192-206 (2007); Barthold et al. J Urol 170, 2396-401 (2003); and Bleyer, W. A. CA Cancer J Clin 40, 355-67 (1990). Fundamentally, because azoospermia results in an inability to reproduce by natural mating, it seems enigmatic as to why this disease remains so prevalent in the human population. Such an epidemiological trend clearly points to the existence of potent environmental factors that disrupt the process of sperm production (i.e. spermatogenesis) or a substantial number of de novo mutations that could arise during a lifetime to render one sterile, but otherwise healthy. See Bhasin et al., J Clin Endocrinol Metab 79, 1525-9 (1994); Bleyer, W. A., CA Cancer J Clin 40, 355-67 (1990); Reijo, R. et al. Nat Genet. 10, 383-93 (1995); Oates et al., Hum Reprod 17, 2813-24 (2002). In fact, this is true in numerous cases, as such de novo mutations account for several types of male-factor infertility already defined at a genetic level and increasing numbers of males are left infertile during their childhood by cancer chemotherapy. See Sadeghi-Nejad, et al., Urol J 4, 192-206 (2007); Reijo et al. Nat Genet. 10, 383-93 (1995); Bleyer et al., CA Cancer J Clin 40, 355-67 (1990); Oates et al., Hum Reprod 17, 2813-24 (2002); Bhasin, S., J Clin Endocrinol Metab 92, 1995-2004 (2007); and Geens, M. et al., Hum Reprod Update 14, 121-30 (2008). As a new hope for many infertile men with azoospermia, a pioneering breakthrough in stem cell biology that manifested strong links between reproductive biology and genetic research was the discovery that mouse testes contained spermatogonial stem cells capable of generating fully functional sperm following isolation and transplantation into testes of another mouse. See Brinster & Zimmermann, Proc Natl Acad Sci USA 91, 11298-302 (1994). Similar experiments soon followed in rats, and isolated mouse spermatogonia were next shown to maintain their regenerative potential after months in culture. See Clouthier et al., Nature 381, 418-21 (1996); Nagano et al., Tissue Cell 30, 389-97 (1998). New culture media supporting the long term proliferation of rodent spermatogonial lines in vitro have since been formulated and scientists are now on the brink of establishing conditions required to cultivate human spermatogonial lines from testis biopsies. See Kanatsu-Shinohara et al., Biol Reprod 69, 612-6 (2003); Hamra, F. K. et al., Proc Natl Acad Sci USA 102, 17430-5 (2005); Conrad, S. et al. Nature (2008); and Kossack, N. et al. “Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells.” Stem Cells (2008). Ostensibly, the ability to propagate spermatogonial lines in culture, prior to using them to produce functional spermatozoa by transplanting them back into the testes of their own donor, presents a clear strategy to cure many existing types of male infertility. Due in large part to the multipotent nature of germline stem cells however, before these breakthroughs are translated into practice it is imperative that preclinical details of such cellular therapies first be stringently evaluated in more advanced, non-human recipients of medical relevance. See Geens, M. et al., Hum Reprod Update 14, 121-30 (2008); Conrad, S. et al. “Generation of pluripotent stem cells from adult human testis.” Nature (2008); Kossack, N. et al. “Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells.” Stem Cells (2008); Hermann, B. P. et al. Stem Cells 25, 2330-8 (2007); and Zhang et al., J Cell Physiol 211, 149-58 (2007).
Production of Transgenic Animals
In mice, embryonic stem (ES) cell-based knockout technology is very efficient for single gene targeting, and it can be combined as well with the usage of random mutagens, such as chemical mutagenic agents, viruses or transposons, for the large-scale generation of ES cell libraries, carrying different molecularly marked knockout alleles. These ES cell clones can be used for the production of knockout mice.
While this methodology is applicable for mice, it cannot be employed with rats or with other laboratory animals. Furthermore, no similar or equivalent techniques to the mouse ES cell technique have yet been developed that would be applicable to a variety of animal models and not limited to one animal species like the ES cell technique in mice. For example, due to the above-mentioned technical limitation very few rat knockout strains are currently existing worldwide. This may at least partially be the result of the practicability of random mutagenesis in animals, which has proven to be questionable for several reasons. For example, the requirement of a large number of offspring, the time for rearing offspring, the costs of establishing and maintaining large-scale animal facilities are some of the factors to be considered when generating transgenic animals using random mutagenesis in animals. Accordingly, there exists a need for more advantageous methods of targeted mutagenesis that can be applied in a variety of animal models and are more practicable.
Current technologies used to create transgenic rats require a high level of expertise and are costly to produce. Additionally, there are many disadvantages to the currently available recipient rat models for testicular transplantation of donor stem cells. These disadvantages include but are not limited to: (1) lower germline transmission from the donor cells to progeny, due to high levels of competition from endogenous sperm cell production; (2) the need for a high number of stems cells to be transplanted into recipient testes to produce transgenic progeny; (3) a large number of progeny must be produced to yield the desired mutant rat line; and (4) the need for a high dose of cytotoxic chemicals or irradiation to achieve effective engraftment of testes by donor stem cells. In conventional protocols, moreover, the most effective levels of stem cell engraftment have not been realized because lethal doses of irradiation or cytotoxic reagents required for effective stem cell engraftment kill the recipients. Finally, production of rat lines with loss or gain of function gene mutations requires tedious, time-consuming and prohibitively expensive micromanipulation of embryos.
There is a general need to annotate the human genome with function, linking laboratory animals into this process is a necessary requirement for accelerating improvements in health care. For example, extensive phenotyping and detailed analysis of inbred animals strains has resulted in the localization of hundreds of loci involved in complex diseases. These “quantitative trait loci” (QTLs) demonstrate genetic linkage to many disease traits which are shared between laboratory animals, such as rats, and humans. Examples for such diseases include hypertension, neuronal regeneration, ischemic cerebrovascular and cardiovascular diseases, and diabetes. See Jacob, H. J. et al., Nature Rev. Genet. 3:33 (2002), and Hubner, N. et al., loc. cit. 37:243 (2005).